NF-κB is a heterodimeric transcription transcription factor regulating the expression of multiple inflammatory genes. The expression of more than 70 known proteins is transcriptionally regulated by the binding of NF-κB to specific sequence elements in the promoter region of these genes (Baeuerle and Baichwal, Advances in Immunology 65:111-137, 1997) NF-κB has been implicated in many pathophysiologic processes including angiogenesis (Koch et al., Nature 376:517-519, 1995), atherosclerosis (Brand et al., J Clin Inv. 97:1715-1722, 1996), endotoxic shock and sepsis (Bohrer et al., J. Clin. Inv. 100:972-985, 1997), inflammatory bowel disease (Panes et al., Am J Physiol. 269:H1955-H1964, 1995), ischemia/reperfusion injury (Zwacka et al., Nature Medicine 4:698-704, 1998), and allergic lung inflammation (Gosset et al., Int Arch Allergy Immunol. 106:69-77, 1995). Because of the central role of NF-κB in inflammatory disease, inhibition of NF-κB by targeting regulatory proteins in the NF-κB activation pathway represents an attractive strategy for generating anti-inflammatory therapeutics.
The IκB kinases (IKKs), are key regulatory signaling molecules coordinating the activation of NF-κB. IKK-1 and IKK-2 are structurally unique kinases containing an N-terminal kinase domain with a dual serine activation loop, a leucine zipper domain, and a C-terminal helix-loop-helix domain and serine cluster. IKK enzymes show relatively low sequence homologies with other kinases, and early profiles with known kinase inhibitors have not identified compounds with striking potency. Kinetic analysis shows that IKK-2 binds to and phosphorylates IκBα, IκBβ, and IKBε with high and relatively equal affinities (Heilker et.al. 1999). Recombinant IKK-2 phosphorylates IκBα peptide 26-42 with near equal affinity to full length IκBα, however the native IKK enzyme complex phosphorylates full length IκBα 25,000 fold more efficiently, suggesting important regulatory sequences in the C-terminal region of IκBα, or additional regulatory proteins in the IKK enzyme complex that accelerate the rate of catalysis (Burke et al., Journal of Biological Chemistry 274:36146-36152, 1999). Phosphorylation of IκBα occurs via a random sequential kinetic mechanism, meaning either ATP or IκBα may bind first to IKK-2, t that both must be bound before phosphorylation of IκBα can take place (Peet and Li, Journal of Biological Chemistry 274:32655-32661, 1999). IKK-2 binds ATP with uniquely high affinity (Ki=130 nM) compared to other serine-threonine kinases such as p38 and JNK perhaps indicating a unique ATP binding pocket that reflects the relatively poor activity to many broad specificity kinase inhibitors when tested against IKK-2. To date, no crystal structure of IKK-2 has been reported. However homology modeling has identified 3 structural domains including an N-terminal kinase domain with an activation loop, a leucine zipper domain that likely mediates the formation of IKK-1 and IKK-2 homo/heterodimers, and a C-terminal helix-loop-helix with serine rich tail. Activation of IKK-2 is critically dependent upon phosphorylation of serine 177 and 181 in the activation or T loop. Alanine mutations abolish activity, while glutamate mutations result in a constitutively active enzyme (Mercurio et al. Science 278:860-866, 1997; Delhase et al., Science 284:30 313, 1999).
IKK-1 and IKK-2 occur both as heterodimers and IKK-2 homodimers, and are associated with a 700-900 kDa cytoplasmic enzyme complex called the “IKK Signalsome” (Mercurio et al., Science 278:860-866, 1997). Another component, IKKAP-1 or NEMO/IKKγ has no apparent catalytic function but will associate directly with IKK-2 and is necessary for full activation of NF-κB (Mercurio et al., Mol Cell Biol. 19:1526-1538, 1999). Many immune and inflammatory mediators including TNFα, lipopolysaccharide (LPS), IL-1, anti-CD28, CD40L, FasL, viral infection, and oxidative stress have been shown to lead to NF-κB activation. Although the receptor complexes that transduce these diverse stimuli appear very different in their protein components, it is understood that each of these stimulation events leads to activation of the IKKs and NF-κB.
The IKK complex appears to be the central integrator of diverse inflammatory signals leading to the phosphorylation of I κB. IKKs are activated at dual serine residues by upstream kinases including NF-κB inducing kinase, NIK (Malinin et al., Nature 385:540-544, 1997), and MEKK-1 (Yujiri et al., Science 282:1911-1914, 1998). The differential activities of NIK and MEKK-1 remain unclear although initial data indicates these kinases may preferentially activate IKK-1 and IKK-2, respectively. Activated IKK phosphorylates a cytoplasmic inhibitor protein, IκB which binds NF-κB, thereby masking a nuclear localization signal present in Rel proteins (Cramer et al., Structure 7:R1-R6, 1999). IKK phosphorylation of IκB on serines 32 and 36 forms a structural motif recognized by the E3 ligase, βTRcP (Yaron et al., Nature 396:590-594, 1998). Docking of βTRcP results in the formation of a ligase complex which polyubiquitinates IκB thus targeting it for degradation by the 26S proteosome. Free NF-κB is then identified by nuclear transport proteins which translocate it to the nucleus where it can associate with sequence specific regulatory elements on gene promoters.
Although both kinases can phosphorylate IκB in vitro, early studies using genetic mutants indicated that IKK-2, but not IKK-1, was essential for activation of NF-κB by pro-inflammatory stimuli such as IL-1β and TNFα. Furthermore, only catalytically inactive mutants of IKK-2 blocked the expression of NF-κB regulated genes such as monocyte chemotactic protein (MCP-1) and intercellular adhesion molecule (ICAM-1) (Mercurio et al, Science 278:860-866, 1997). Studies of knockout animals for IKK-1 and IKK-2 substantiate these initial findings (Hu et al., Science 284:316-320, 1999; Li et al., Genes & Development 13:1322-1328, 1999; Li et al., Science 284:321-324, 1999; Takeda et al., Science 84:313-316, 1999; Tanaka et al., Immunity 10:421-429, 1999). IKK-1−/− animals were born alive but died within hours. Pups showed abnormalities of the skin due to defective proliferation and differentiation, but showed no gross deficiency in cytokine induced activation of NF-κB. In contrast, IKK-2−/− embryos died at day 14-16 of pregnancy from liver degeneration and apoptosis that bore a striking resemblance to that observed in Rel A knock-out animals (Beg et al., Nature 376:167-170, 1995). Furthermore, embryonic fibroblasts from IKK-2−/− animals exhibited markedly reduced NF-κB activation following cytokine stimulation, while IKK-1−/− did not.
Accordingly, cell and animal experiments indicate that IKK-2 is a central regulator of the pro-inflammatory role of NF-κB. IKK-2 is activated in response to multiple inflammatory stimuli and signaling pathways, many of which play an important role in respiratory disease including IL-1β, LPS, TNFα, CD3/CD28 (antigen presentation), CD40L, viral infection, and oxidative stress. The ubiquitous expression of NF-κB, along with its response to multiple stimuli means that almost all cell types present in the lung are potential target for anti-NF-κB/IKK-2 therapy. This includes alveolar epithelium, mast cells, fibroblasts, vascular endothelium, and infiltrating leukocytes; neutrophils, macrophages, lympophocytes, eosinophils and basophils. By inhibiting the expression of genes such as cyclooxygenase-2 and 12-lipoxygenase (synthesis of inflammatory mediators), TAP-1 peptide transporter (antigen processing), MHC class I H-2K and class II invariant chains (antigen presentation), E-selectin and vascular cell adhesion molecule (leukocyte recruitment), interleukins-1, 2, 6, 8 (cytokines), RANTES, eotaxin, GM-CSF (chemokines), and superoxide dismutase and NADPH quinone oxidoreductase (reactive oxygen species), inhibitors of IKK-2 are believed to display broad anti-inflammatory activity.
International Publication No. WO 98/18782 to Celltech Therapeutics Limited discloses 4-pyridyl pyrimidine compounds which are allegedly useful in the prophylaxis and treatment of immune diseases, allergic diseases involving mast cells or eosinophils, and diseases involving inappropriate platelet activation.
Accordingly, there is a need in the art for selective inhibitors of IKK, particularly IKK2 inhibitors. In addition, there is a need for pharmaceutical compositions comprising one or more inhibitors, as well as to methods for treating conditions in animals which are responsive to such inhibitors. The present invention fulfills these needs, and provides further related advantages.
Citation of identification of any reference in Section 2 of this application shall not be construed as an admission that such reference is prior art to the present invention.